|
(11) | EP 1 958 643 A2 |
| (12) | EUROPEAN PATENT APPLICATION |
| published in accordance with Art. 158(3) EPC |
|
|
|
|
|||||||||||||||||||
| (54) | STABLE FORMULATIONS CONTAINING ENHANCING PROPORTIONS OF GAMMA- AND ALPHA-INTERFERONS |
| (57) The present invention relates to stable pharmaceutical formulations to be applied
by parenteral (liquids or freeze-dried), or topical way (gel, unguent or cream) that
contain different quantities of the recombinant interferons gamma and alpha in synergistic proportions for the treatment of pathological
events that contemplate the malignant or benign not-physiological growth of cells in tissue or organs. |
Technical Field of
[0001] The present invention relates to biotechnology and medical sciences, particularly to stabilized pharmaceutical formulations that contain recombinant interferons gamma and alpha in synergistic proportions for the inhibition of the cell growth in different tissues or organs of human beings.
State of the art
[0002] The variety of effects of the interferons type I (in English "Interferons", shortened IFNs) creates a great therapeutic potential for their applications. The application of IFNs is beneficial in the treatment of various types of cancer, among them are included leukemias (US 5830455), basal cell carcinoma (US 5028422), squamous cell carcinoma (US 5256410), breast cancer (US 5024833), gastrointestinal tumors (US 5444064; 5814640), and actinic keratosis (US 5002764). Different cell types show a differential sensitivity to the IFNs, and the concentrations to inhibit their growth can vary over a wide range (Borden E., et al. (1981) Progress in Hematology. vol XII, Brown EB., editor, 299-339), which exemplifies the differences in their capacity to inhibit the cell growth (Dahl H. (1983). Human interferon and cell growth inhibition. VII. Reversibility of interferon activities. J Interferon Animal, 3:327-332; Willson J.K.V., Bittner G., et al. (1984) Antiproliferative activity of human interferons against ovarian cancer cells grown in human tumor stem cell assay. J Interferon Animal, 4:441-447; Hu R., Gan Y., et al. (1993) Evidence for multiple binding sites for several components of human lymphoblastoid interferon-alpha. J Biol Chem, 268:12591-12595), and to the activity antitumoral (Quesada JR., Talpaz M., et al. (1986) Clinical toxicity of interferons in cancer patients: to review. J Clin Oncol, 4:234-243).
[0003] The use of the IFNs in the cancer therapy has not satisfied the expectations from the in vitro studies and the properties of these powerful biological molecules possess. Different therapeutic schedules have been tested without clear beneficial effects and impact (Strander H., and Oberg K., (1992) Clinical use of interferons. Solid tumors INTERFERON. Principles and Medical Applications. Publishing Baron S., Coppenhaver DH., Dianzani F., Fleischmann WR., Jr. Hughes TK., Jr. Klimpel GR., Niesel DW., Staton GJ., and Tyring SK., 533-561).
[0004] In an effort to reach better effects in the therapies, the IFNs were employed at high doses, but the beneficial potential expected response does not appear, owed to various factors, among them the adverse reactions that are produced with said doses (Lane H. C. (1990) Interferon-alpha in patients with asymptomatic human immunodeficiency virus (HIV) infection. A randomized, placebo-controlled trial. Annals of internal Medicate, 112:805-811).
[0005] In addition, the IFNs have been used in combined form exploiting their synergistic effects. The combination of IFN alpha and IFN gamma has been described in in vitro studies with cultures from keloids fibroblast (Tredget EE., Wang R., et al. (2000) Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo. J Interferon Cytokine Animal, 20:143-151). In this work, the combined utilization of the IFNs alpha and gamma is mentioned, but the resulted data come from in vitro and in cells originating from keloids from children. These authors did not carry out any clinical trial and did not evaluate the effect of the IFNs combination on cells of adults keloids that are poor responder to the interferons.
[0006] The patent EP 0107498 shows the combination of the interferons alpha and gamma in the cell line of melanoma Hs294T, but dose not describe this effect in other types of cells like primary culture of basal cell carcinoma, or of a glioblastoma (GL-5), or of a laryngeal carcinoma (HEp-2).
[0007] The alternated utilization of natural IFN alpha and recombinant IFN gamma has also been described for the treatment of renal and lung metastasis (Fujii A., Yui-In K., et al. (1999) Preliminary results of the alternating administration of natural interferon-alpha and recombinant interferon-gamma for metastasic renal cell carcinoma BJU Int.; 84:399-404). The combination of IFN alpha 2, or alpha 4 or the hybrid delta 4 alpha 2 Bgl II alpha 1 with IFN gamma was described in the cell lines RT4 (bladder carcinoma) and in A2182 (lung adenocarcinoma), and possesses a superior antiproliferative effect than IFNs type I or IFN gamma alone, (Hubbell H.R., Craft J.TO., et al. (1987) Synergistic antiproliferative effect of recombinant alpha-interferons with recombinant gamma-interferon. J Biol Response Mod, 6:141-153). A synergistic effect among the IFN gamma (1000 IU/mL) and the IFN alpha 2 (1000 IU/mL) was shown in the cell line A459 (alveolar tumor), (Martyre M. C., Beaupain R., et al. (1987) Potentiation of antiproliferative activity by mix of human recombinant IFN-alpha 2 and -gamma on growth of human cancer nodules maintained in continuous organotypic culture. Eur J Cancer Clin Oncol, 23:917-920), as well as in cell lines established from non-small cell lung anaplastic carcinoma (Hand A., Pelin K., et al. (1993) Interferon-alpha and interferon-gamma combined with chemotherapy: in vitro sensitivity studies in non-small cell lung-cancer cell lines. Anticancer Drugs, 4:365-368).
[0008] The combination of IFN alpha and IFN gamma has been described in studies with the cell line HepG2, (Mizukoshi E., Kaneko S., et al. (1999) Up-regulation of type I interferon receptor by IFN-gamma. J Interferon Cytokine Animal, 19:1019-1023) and in the cell line AVA5 (Okuse C., Rinaudo J. A., et al. (2005) Enhancement of antiviral activity against hepatitis C virus in vitro by interferon combination therapy. Antiviral Animal, 65:23-34). These authors do not determine the antiproliferative effect, neither the more effective proportions in the combination of the alpha and gamma interferons in the cell line HepG2. In addition, the synergistic effect has been explored for TNF alpha and IFN gamma in the cell line Hepa1-6, a murine hepatoma (Sasagawa T., Hlaing M., et al. (2000) Synergistic induction of apoptosis in murine hepatoma Hepa1-6 cells by IFN-GAMMA and TNF-alpha. Biochem Biophys Common Animal, 272:674-680).
[0009] In the US patent 5190751 the inhibition of the growth of leukemias cell lines of type B and of type T by the combination of IFN alpha and gamma is described. In none of the T cell lines evaluated, the potentiation of the growth inhibitory effect was observed, and in certain experimental conditions, the effects of the combinations were antagonistic. In the patent EP 010749 and in a publication (Czarniecki C. W., Fennie C. W., et al. (1984) Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons. J Virol. 49:490-496), is shown also that the combination of the IFNs alpha and gamma not always is synergic and can be antagonistic. The efficacy of the combinations in a very extensive range is mentioned, but it is not shown.
[0010] These data indicates that the employment of combinations of IFN alpha and gamma should be evaluated for an experimental definition which permit to identify what condition is the favorable one to establish an optimum combination for the treatment of an inadequate cell growth in a given tissues or organs. For such reason, to support a therapy and adequate dose these should be evaluated in experiments in vitro and in controlled clinical trials.
[0011] In a study with cell lines of Gliomas, the IFN gamma affected the characteristics of malignancy such as the proliferation and the migration of the studied tumor cells (Knupfer M. M., Knupfer H., et al. (2001) Interferon-gamma inhibits growth and migration of A172 human glioblastoma cells. Anticancer Animal, 21:3989-3994). Other wise, negative results with the employment of IFN gamma to treat the gliomas have been reported (Mahaley M. S., Bertsch L., Jr. et al. (1988) Systemic gamma-interferon therapy for recurrent gliomas. J Neurosurg, 69:826-829). The simultaneous employment of IFN gamma and IFN beta has turned out to be efficient in the inhibition of the growth of the cell line GBM-18, a multidrug resistant astrocitoma (Reddy P. G., et al. (1991) Systemic gamma-interferon therapy for recurrent gliomas. J Natl Cancer Inst, 83:1307-1315). Besides, it has been described, the combination of IFN gamma with alpha-difluoromethylornitine (DFMO) for the treatment of these tumors (US 4499072). The US patent 5002879, describes a similar therapy utilizing DFMO next to killer cells activated by lymphokines and IL-2. With respect to IFN alpha, its combination with other drugs has had not favorable effects in the treatment of the gliomas, and has shown toxicity (Buckner J. C., Burch P. A., et al (1998) Phase II trial of recombinant interferon-alpha-2a and eflornithine in patients with recurrent glioma. J Neurooncol. 36:65-70; Chang S. M., Barker F. G., et al. (1998) High dose oral tamoxifen and subcutaneous interferon alpha-2a for recurrent glioma. J Neurooncol, 37:169-176). Then, the treatment of this type of tumor can be favored for the combined use of the IFN alpha and the IFN gamma, on the base of an adequate selection of the proportions of its combination based on in vitro experiments and in clinical trials.
[0012] The larynx is the second more frequent place of cancer of the upper aero-digestive tract after the oral cavity. The laryngeal carcinoma is the most frequent tumor of head and neck and the most common cancer of larynx is the squamous cell carcinoma of (95% of all the cases). The survival in the cases of laryngeal tumors T3 and T4 is only 5 years in approximately the 30% of the patients submitted to laryngectomia (Djordjevic V., Milovanovic J., et al. (2004) Radical surgery of the malignant laryngeal tumors. Minutes Chir Lugosl, 51:31-35). It has been shown that the radiation therapy and the chemotherapy are not effective for the treatment of this carcinoma (Chen W., Guo X., et al. (2004) Long-term follow-up observation of clinical therapy for laryngeal carcinoma recurrence and cervical metastasis Lin Chuang Er Bi Yan Hou Ke Za Zhi. 18:536-537).
[0013] Nevertheless, the polychemotherapy together with the employment of IFN alpha has turned out beneficial in the treatment of the laryngeal cancer (Mantz C. A., Vokes E. E., (2001) Sequential induction chemotherapy and concomitant chemoradiotherapy in the management of locoregionally advanced laryngeal cancer Ann Oncol, 12:343-347). The combination of IL-2 and IFN alpha was evaluated in a phase II trial as therapy for the laryngeal carcinoma, but the results were not satisfactory (Clayman G. L., Young G., et al. (1992) Detection of regulatory factors of lymphokine-activated killer cell activity in head and neck cancer patients treated with interleukin-2 and interferon alpha. Ann Otol Rhinol Laryngol, 101:909-915). Few advances exist in the therapeutic of the laryngeal tumors. The combined use of the IFNs alpha and gamma would be able to contribute to improve the existing therapies to fight this type of tumors.
[0014] The US patent 5503828 describes a composition of interferons characterized by containing at less 50% of the alleles of IFN alpha 2 and IFN alpha 8 and one or more additional species of IFNs of a group formed by IFN alpha 4, alpha 7, alpha 10 alpha 16, alpha 17, and alpha 21. While, the US patent 4503035 shows a preparation of some species of IFN alpha, but that does not include alpha 1, alpha 5, alpha 14, and al IFN omega. These patents do not describe a formulation formed by the combination of recombinants IFN gamma and IFN alpha 2.
[0015] The US patent 5762923 details an interferon liquid composition diluted in water with a not ionic detergent and benzilic alcohol in sufficient quantities to stabilize the IFN alpha that contains besides, an acid buffer. On the other hand, the US patent 4847079 describes a pharmaceutical composition of interferon and timerosal, while the US patent 4675184 shows an interferon formulation with polyhydric alcohol and an organic buffer as stabilizer and a conventional carrier or diluents of pH 3-6. The composition can have additionally an anionic surfactant and/or albumin as stabilizer. In the patents US 5236707 and US 5431909 are described amines as stabilizers (aliphatic primary amines) and organic sales of lithium, that protect the interferon from degradation and stabilize it.
[0016] The US patent 4496537 refers liquids stable formulations of interferon-alpha that include human serum albumin composition, and alanine or glycine, water and a buffer system capable to maintain the pH between 6.5 and 8.0.
[0017] The US patent 5935566 describes stable formulations of interferon-alpha that include in their composition a buffer system capable to maintain the pH in the range from 4.5 to 7.1, polysorbate 80 as stabilizer, EDTA as chelating agent, sodium chloride as isotonizing agent, and m-cresol as antimicrobial preserving.
[0018] The US patent 0170207 describes stable formulations of interferon-alpha that include in their composition a buffer system capable to maintain the pH in the range from 4.5 to 9.0, a stabilizing agent, a not ionic surfactant and a regulator of the osmotic pressure.
[0019] In the application WO 89/04177 liquids pharmaceutical formulations of interferon-gamma are described that contains a buffer solution that maintains the pH in the range from 4.0 to 6.0, a polyhidroxyl sugar as stabilizer and a not ionic detergent. The US patent 4895716 refers to compositions and methods for the stabilization of the interferon-gamma with lactobionic acid in a buffer glycine/acetate solution.
[0020] The US patent 5676942 describes pharmaceutical compositions formed by subtypes of interferons of type I obtained from natural sources, but not combined with interferon gamma and do not define the proportions of those combinations, only describes those combinations for viral infections and not for the treatment of tumors. None of the previously described reports has utilized, characterized or mentioned a pharmaceutical formulation that contain the recombinant IFNs gamma and alpha 2 together in synergistic combinations. Potentialities in the combined utilization exist for IFN gamma and IFNs type I when they are mixed in defined proportions for the treatment of cell growth having varying degrees of resistance to established therapies and/or combinations thereof.
[0021] Keeping in mind these premises, it is necessary to develop stable pharmaceutical formulations that contain these IFNs in proportions that permit their wider, simple, efficient, and safe use, in individuals with benign or malignant tissue formations in need of thereof. This will permit an optimized management of the combinations and provides a more successful use of the therapeutic in patients in need of this treatment.
Explanation of the invention.
[0022] The present invention resolves the problem before mentioned, providing stable pharmaceutical formulations to be applied by parenteral (liquids or freeze-dried), or topical way (gel, ungent or cream). They contain different quantities of the recombinant interferons gamma and alpha in synergistic proportions for the treatment of pathological events that contemplate not physiological benign or malignant growth of tissue or organs and that contain besides, excipients or vehicles pharmaceutically acceptable.
[0023] These formulations are the result of the in vitro assays with cell lines of different sensibility to IFNs and of clinical trials in different tumor entities, as well as of the evaluation of biological and physical-chemical stability of the recombinant IFNs gamma and alpha 2 in the presence of the different excipients or vehicles pharmaceutically acceptable.
[0024] The freeze-dried stable pharmaceutical formulations are composed of the recombinant IFN gamma and alpha 2 mixed in a buffer solution capable to maintain the pH between 4.9 and 7.5, which can be the ammonium or sodium acetate, the sodium succinate, sodium and/or potassium phosphate or the sodium citrate/phosphate.
[0025] These formulations also are composed of at least of a component selected from not reducing sugars compounds, amino acids, surfactants and stabilizing polymers. The not reducing sugars can be the saccharose or threhalose; the amino acids can be glycine, histidine or leucine; while as surfactants are described polysorbate 20 or the polysorbate 80 and like stabilizing polymer polyethylene glycol, dextran or hydroxyethyl starch.
[0026] A materialization of the invention defined that the buffer solution should be employed in a range of concentration between 10 and 20 mM. The saccharose or threhalose, should be used between 5 and 100 mg/mL; glycine, histidine or leucine should be employed in a range of concentration between 1 and 20 mg/mL. The polysorbate should be employed between 0, 01 and 1 mg/mL, while polyetilenglycol, dextran, and hydroxyethyl starch, they are employed in a range of concentration between 5 and 50 mg/mL.
[0027] Several materializations of the invention describe freeze-dried stable pharmaceutical formulations that contain recombinant IFN gamma in a range of concentration between 5.6 x 108 IU and 1.4 x 108 IU and recombinant IFN alpha 2 in a range of concentration between 6.8 x 108 IU and 1.7 x 108 IU. Or recombinant IFN gamma in a range of concentration between 2.0 x 108 IU and 0.5 x 108 IU and recombinant IFN alpha 2 in a range of concentration between 12.0 x 108 IU and 3.0 x 108 IU. Or recombinant IFN gamma in a range of concentration between 4.0 x 108 IU and 1.0 x 108 IU and recombinant IFN alpha 2 in a range of concentration between 80 x 108 IU and 20 x 108 UI. The formulations contain additionally 0.0802 g of potassium di-hydrogen phosphate, 0.249 g di-hydrated di-sodium hydrogenphosphate, 4 g of saccharose, 0.8 g of glycine, 0.03 g of Tween 20,1 g of polyetilenglycol 6000, and water for injection sufficient quantity for 100 mL and for 0.5 mL, 1 mL, 5 mL and 10 mL in the respective equivalent proportions.
[0028] The definition to mix the recombinant IFN gamma and IFN alpha in a range of defined combination was obtained after an isobologram analysis. The concentration of recombinant IFN gamma between 5.6 x 108 IU and 1.4 x 108 IU and recombinant IFN alpha 2 in a range of concentration between 6.8 x 108 IU and 1.7 x 108 IU, in one of the freeze-dried stable pharmaceutical formulations, was reached from the analysis of the studies of the inhibition of the growth of the primary culture originating from keloids (Kel 5a, Kel 17a) and from the CBC III. After an isobologram analysis the combination of 100 IU/mL (10 ng/mL) for recombinant IFN gamma with 100 IU/mL (0.5 ng/mL) of recombinant IFN alpha 2b that reduce the cell growth in vitro in a 21 %, 43% and 47%, respectively, was identified (to see examples 1, 2 and 3, figures 1, Table 1).
[0029] The mixture of recombinant IFN gamma in a range of concentration between 2.0 x 108 IU and 0.5 x 108 IU and recombinant IFN alpha 2 in a range of concentration between 12.0 x 108 IU and 3.0 x 108 IU for the formulation was defined utilizing a clinical trial and report of treated cases by compassion. The randomized, controlled, triple blind clinical trial, evaluated the efficacy of the intralesional (I.L.) treatment in patients with basal cell carcinoma utilizing the stable freeze-dried formulation defined above (to see example 7, tables 9, 10, 11, and 12).
[0030] In the report of treated cases by compassion, that also contributed to define these proportions, were treated patients with epidermoid carcinoma (patient 1) and a patient with multiple recurrent basal cell carcinomas, and with previous grafts (patient 2), (to see example 8 figures 5 to, b, c, d; patient 1, and figures 6 to, b, c; patient 2, respectively).
[0031] The formulation that contains recombinant IFN gamma in a range of concentration between 4.0 x 108 IU and 1.0 x 108 IU and recombinant IFN alpha 2 in a range of concentration between 80 x 108 IU and 20 x 108 IU was defined with the analysis of the results from the study of the inhibition of the growth of glioblastoma (GL-5) cells by 50 IU/mL (5 ng/mL) of recombinant IFN gamma with 100 IU/mL (0.5 ng/mL) of recombinant IFN alpha 2b. In this way, an inhibition of the growth of the 55% is reached (example 3). In addition, it was taken into account the analysis of the study with the cell line HEp-2. In this case, the quantities of IFNs are of 5 IU/mL (0.5 ng/mL) of recombinant IFN gamma with 75 IU/mL (0.375 ng/mL) of recombinant IFN alpha 2b. With that, optimum combination is reached to reduce the cell growth in vitro in a 76% (to see examples 1, 2 and 3).
[0032] In addition were developed pharmaceutical stable liquid formulations. In these formulations the proportions of the recombinant IFNs gamma and alpha, were maintained as described for the freeze-dried formulations, but their pharmaceutical ingredients varied to achieve a greater stability to these mixtures of the IFNs.
[0033] As a consequence of this work a materialization of the invention describes liquid stable pharmaceutical formulations that contain a buffer solution and at least a component selected from non-reducing sugars, amino acids, surfactants, stabilizing polymers antioxidant/chelating components and isotonizing agents. These formulations employ a water based solvent that can contain or not preserving agents just as the mixture of methyl- and propyl-paraben.
[0034] Another materialization of the invention situates the definition of liquid stable pharmaceutical formulations that employ a buffer solution capable to maintain the pH between 4.9 and 6.5. This buffer can be ammonium or sodium acetate, sodium succinate, sodium and/or potassium phosphate, citrate/phosphate. These formulations can employ as surfactants polysorbate 20 or polysorbate 80; as antioxidant/chelating agent EDTA or acetyl-cysteine; while as amino acids can be used histidine, L-arginine, L-alanine, glycine or lysine. As stabilizing polymer is defined the utilization of hydroxyethyl starch or dextran and as isotonizing agent sodium chloride, potassium chloride, propylene glycol, mannitol, glycerol, saccharose or threhalose.
[0035] A materialization of the invention collects that the liquid stable pharmaceutical formulations employ a buffer solution in a range of concentration between 10 and 100 mM. In this formulation the polysorbate is employed in a range of concentration between 0, 01 and 1 mg/mL; the EDTA or the acetyl-cysteine are employed in a range of concentration between 0, 01 and 1 mg/mL. The amino acids histidine, L-arginine, L-alanine, glycine or lysine are at a concentration between 1 and 20 mg/mL; the hidroxietil starch and dextran are employed in a range of concentration between 5 and 50 mg/mL and the isotonizing agents are found in sufficient quantity to do isotonic the solution.
[0036] Other materializations explain the quantities of all the pharmaceutical ingredients of the liquid stable pharmaceutical formulations necessary for the physico-chemical and biological stability of the mixtures of the recombinant IFNs gamma and alpha described previously. These liquid formulations contain besides the IFNs, 0.708 g of sodium acetate, 0.079 mL of acetic acid, 0.01 g of Tween 20, 5 g of manitol, and water for injection sufficient quantity for 100 mL and for 0.5 mL, 1 mL, 5 mL and 10 mL in the respective equivalent proportions.
[0037] This invention defines the proportions of mixtures of IFNs gamma and alpha that can be profitable for the treatment of the benign or malignant overgrowth of cells. This will permit to employ smaller dose, less time of treatment and to maintain the same therapeutic effects or to achieve effects over the ones that have been reached until today with the employment of the interferons in the treatment of the tumors or other aberrant events of cell growth. Lowering the dose will permit to expect less adverse effects or smaller intensity of them, that will give a better quality of life to the patients and will permit them to obtain the benefits of the use of these powerful drugs.
[0038] The invention defines formulations of the mixture of recombinant IFN gamma and IFN alpha 2 that have not been described previously, that facilitate the management and clinical use of this therapeutic combination and their commercialization.
[0039] The freeze-dried and liquid stable pharmaceutical formulations that contain mixtures of the recombinant IFNs gamma and alpha 2 in synergistic proportions for the inhibition of the proliferation described in the invention, has an extensive spectrum of clinical use. It is shown in vivo utilizing these formulations, that in important oncological diseases, the combination of the recombinant IFN gamma and the IFN alpha 2 is effective utilized simultaneous and intratumoral.
[0040] This combination is capable of having equals curative effects on tumors in shorter time and with a higher esthetic effect when compared with that obtained for its separated components. The use of these combinations will permit to include greater therapeutic possibilities to fight the cancer. This it is collected in a materialization of the invention where is exposed that the freeze-dried or liquid formulations can be employed in the treatment of solid benign or malignant tumors, utilized in independent forms or combined with chemotherapy, radiation therapy or the combination of both.
[0041] The utilization of these formulations in combination with other therapeutic agents is supported in the results obtained with the treatment of a patient with a giant basal cell tumor with the combination of recombinant IFN gamma and IFN alpha 2 along with cisplatin (to see example 10 and figure 9).
[0042] It is described in the invention how the combined employment of the interferons gamma and alpha 2 permits to reduce and/or to cure tumors of very badly forecast and of distorting esthetic effects.
[0043] According to the characteristics of several benign and ontological entities where predominates an uncontrolled growth of cells, they can be susceptible to be treated with these formulations. Among them are: Tumors of the cells from hematopoyetic tissue such as the acute or chronic myeloid leukemia, acute or chronic lymphocytic leukemia, as well as the leukemias of T, or B cells and the lymphoma of the central nervous system. Also can be treated laryngeal carcinomas, laryngeal papilomatosis, lipoma, epidermoid and intradermal cyst, liposarcoma, neurofibroma, and sebaceous hyperplasia. Can be beneficed with the use of these pharmaceutical formulations tumors from peripheral and central nervous system as astrocitomas, multiform glioblastomas, ependymomas, ganglioneuromas, pilocitic astrocitomas, mixed gliomas, oligodendrogliomas, gliomas of the optic nerve, primitive neuroectodermal tumors, acoustic neuromas, cordomas, craniopharyngioma, medulloblastoma, meningiomas, neurofibromatosis, pseudotumors of brain, tuberose sclerosis, metastasic cerebral tumors. Other susceptible tumors to be treated are the cavernous hemangiomas, hepatocellular adenomas, focal hyperplasia nodular, pineal tumors, pituitary adenomas, vascular tumors, meningeal carcinomatosis, cherry like angiomas, sebaceous gland hyperplasia. The treatment of tumors of skin as the basal cell carcinoma, squamous cells carcinoma, the dermatofibroma, the piogenic granuloma, skin nevus, as well as seborreic and actinic keratosis can benefit from the therapy with these pharmaceutical formulations.
[0044] Another materialization of the invention describes that these formulations can also be employed for the treatment of proliferative events as fibrosis, dysplasia and hyperplasia.
[0045] According to the results of the clinical trials carried out and described in the examples 7, 8, and 10 as materialization of the invention are defined the intramuscular, intratumoral, and perilesional ways of application of the formulations.
[0046] Other materializations describe the application of topical stable pharmaceutical formulations that contain IFN gamma in a range of concentration between 0.32 x 106 IU and 0.08 x 106 IU and al IFN alpha 2 in a range of concentration between 2.0 x 106 IU and 0.5 x 106 IU by gram of semisolid. The formulations are cream composed by 2.2% IFN gamma, 0.58% IFN alpha, 4% of celtilic alcohol, 10% petroleum jelly (Vaseline®), 2% Tween 60, and 0.2% methylparabeno, propylparabeno. In addition, the composition of unguent was defined by 2.2% IFN gamma 0.58% IFN alpha, 60% of white solid petrolatum, 10% of heavy liquid petrolatum, 3% of span 20, and 0.2% methylparaben and propylparaben. Finally, the gel formulation is composed by 2.2% IFN gamma, 0.58% IFN alpha, 0.5% of Carbopol 940, 0.2% of methylparaben and propylparaben, 0.2% of sodium hydroxide, 0.01% of calcium disodium ethylene diamine tetra-acetate, and 2% ethanol.
[0047] All these formulations are resistant to the fluctuations of temperature, which is profitable for the production of the product, its transportation and storage. They prevent the aggregation of the interferons and therefore they present smaller risk to result immunogenic during the use prolonging of the product. The formulations of semisolid permit the employment by the own patients by not invasive and safe form.
[0048] As another materialization of the invention was defined the employment of these topical stable formulations in the treatment of solid benign or malignant tumors of the skin or mucous membranes, utilized in independent forms or combined with chemotherapy, radiation therapy or the combination of both.
[0049] Another materialization of the invention describes that, the topical stable pharmaceutical formulations can be employed for the treatment of lipoma, epidermoid and intradermal cyst, liposarcoma, neurofibroma, sebaceous hyperplasia, hemangiomas, focal nodular hyperplasia, ependymomas, ganglioneuromas, pilocitic astrocitomas, meningiomas, pineal tumors, pituitary adenomas, vascular tumors, meningeal carcinomatosis, neurofibromatosis, cherry like angiomas, hyperplasia of the sebaceous glands, basal cell carcinoma, squanous cell carcinoma, dermatofibroma, piogenic granuloma, dermal nevus, seborreic and actinic keratosis, and condylomas.
[0050] Another materialization of the invention described the conformation of a kit that contains a vial of recombinant IFN gamma, a vial of recombinant IFN alpha to the concentrations and relations described previously, with a sufficient quantity of water for injection vials, for the dilution and/or dissolution of the IFNs. The kit contains the syringes and adequate needles for the simultaneous administration of the iFNs, previously mixed in one of the vials that contain one of the IFNs.
Brief description of the figures:
[0051]
Figure 1. Growth inhibition of fibroblast primary cell culture originating from biopsies of adult patients with keloids by 1000 IU/mL of recombinant IFN gamma or IFN alpha 2.
Figure 2. Isobologram of the cell growth inhibition by the combination of recombinant IFN gamma and IFN alpha 2b on fibroblasts primary cell culture from keloid (Kel5a).
Figure 3. Isobologram of the cell growth inhibition by the combination of recombinant IFN gamma and IFN alpha 2b on fibroblasts primary cell culture from keloid (Kel17a).
Figures 4. Isobologram of the cell growth inhibition by the combination of recombinant IFN gamma and IFN alpha 2b on fibroblasts primary cell culture from basal cell carcinoma (CBC III).
Figures 5. Isobologram of the cell growth inhibition by the combination of recombinant IFN gamma and IFN alpha 2b on the cell line of the glioblastoma GL-5.
Figure 6. Isobologram of the cell growth inhibition by the combination of recombinant IFN gamma and IFN alpha 2b on the cell line from laryngeal HEp-2.
Figure 7. Patient with epidermoid carcinoma treated with the combination of recombinant IFN gamma and IFN alpha 2b.
Figure 8. Patient with recurrent basal cell carcinoma treated with the combination of recombinant IFN gamma and IFN alpha 2b.
Figure 9. Patient with recurrent basal cell carcinoma treated with the combination of recombinant IFN gamma and IFN alpha 2b and cisplatin. A: before treatment, B: after 1 year of treatment.
Detailed exposition of ways of execution /Examples
Example 1. Inhibition of the cell growth by the recombinant IFNs gamma or alpha on primary cell culture.
[0052] The skin biopsies were obtained from normal skin and from patients that developed basal cell carcinoma or keloids, the last due to damage by surgery or burns. The tissue sample was placed immediately in medium DMEM and fragmented to obtain primary culture by explant method. For the evaluation of the antiproliferative effect of the recombinant IFNs gamma and alpha the following primary culture were evaluated: Fibroblast primary culture (CPF) from keloids (1, 2, 5, 7, 8, 15, 17, 19, 20, 24, 26, 27, 31, 32), CPF from basal cell carcinoma (CBC III) and CPF from normal skin (FibN3 and FibN5). The CPF were growth in a culture media mixture RPMI-1640/DMEM that contained gentamicine (50 µ g/ml), and 12% of calf bovine serum (CBS). All the cultures were incubated at 37°C in a CO2 incubator with 5% humidity. To determine the antiproliferative effect of the IFNs, the cells were seeded at 5 x 104 cells/mL in 96 microwell plates. They were synchronized by changing of fresh medium after 24 hours after seeding. At the end of 96 hours of incubation in the presence of different concentrations of the IFNs was determined the viability of 3 replicas of evaluated experimental conditions utilizing the method crystal violet staining, measuring the absorbance at 580 nm and utilizing a reader plates. The results were defined as the % of growth based on the count of viable cells:
AT72h = Absorbance of cell treated 72h.
AC72h= Absorbance of control cells treated 72h.
AC0h = Absorbance of cell before been treated with IFN.
In the figure 1 is shown the antiproliferative action of the recombinant IFNs gamma or alpha on the growth of the keloids CPF. As it can be observed the IFN gamma or alpha 2b inhibits the cell proliferation in various primary culture, while in other they stimulate their growth. As controls were evaluated the primary culture FibN3 and FibN5, as well as primary culture from biopsy of a CBCIII, and HEp-2, U1752 and GL-5 cell lines.Example 2: Inhibition of the cell growth by the recombinant IFNs gamma or alpha recombinantes on established cell lines.
[0053] The human cell lines studied were: Jurkat (ATCC, TIB-152), GL-5 (Perea S. and., et al. (1993) Minutes Cient Venez, 44:22-27), HEp-2 (ATCC, CCL23). The cells GL-5 were cultured in DMEM, and the HEp-2 in MEM-CANE containing gentamicine (50 µ g/ml) and 10% CBS. The Jurkat cells were incubated in the RPMI medium with gentamicine and 10% CBS. All the culture were incubated at 37°C in a CO2 incubator of with 5% of humidity. To evaluate the antiproliferative effect on GL-5 and HEp-2 cells were seeded at 3 x 104 cells/mL. In the case of the Jurkat cells, these were seeded to 105 cells /mL. After 72 hours of incubation in the presence of different concentrations of the IFNs the viability of 3 replicas were evaluated, utilizing the method of violet crystal staining, and measuring the absorbance at 580 nm and utilizing a reader plates. The results were defined as the % of growth based on the count of viable cells as described in the example 1. As it is observed in the table 1 and in the figure 1, the cell lines HEp-2 (laryngeal carcinoma) and the GL-5 (from a glioblastoma), are very sensitive to IFN gamma and not to IFN alpha.
[0054] In the Table 1 is observed that the line HepG2 (Hepatoma) is not sensitive to these IFNs and that in the cell line Jurkat (lymphoma T), the IFN alpha is the most effective one, result that coincides with the successful employment of the IFN alpha 2 in the treatment of tumor from lymphoid tissue.
Example 3: Combinations of the recombinant IFNs gamma and alpha with more effective antiproliferativa action on the primary culture and cell lines.
[0055] Utilizing the CBC-III and keloids (Kel-5a and Kel-17a) CPF and the cell lines HEp-2 and GL-5, studies of combinations were carried out with recombinant IFNs gamma and alpha 2b, to define optimum mixture with synergistic activity of the inhibition of the cell growth. The data obtained in the studies were analyzed building isobolograms. From the isobolograms studies of CPF originating from biopsies of adult keloids (kel 5a and kel 17a) was defined that the optimum synergistic combination for the inhibition of the growth should be composed of 100 IU/mL (10 ng/mL) of IFN gamma and 100 IU/mL (0. 5 ng/mL) of IFN alpha 2b. With that combination the cell growth is reduced in vitro in a 21% (Kel 5a) and in a 43% (kel 17a) (figures 2 and 3).
[0056] In the isobologram of the figure 4 is shown that the combination of 100 IU/mL (10 ng/mL) of IFN gamma with 100 IU/mL (0. 5 ng/mL) of IFN alpha 2b is synergistic and is the most efficient in reducing the in vitro cell growth of the CBC III in a 47%.
[0057] According to the isobologram that is shown in the figure 5, the optimum synergistic combination to inhibit the growth of the cells of the GL-5 is 50 IU/mL (3 ng/mL) of IFN gamma with 600 IU/mL (5 ng/mL) of IFN alpha 2b. With that combination, the in vitro cell growth is reduced in a 55%.
[0058] In the isobologram represented in the figure 6 is shown the optimum synergistic combination of IFN gamma and alpha to obtain the best antiproliferative effect on the HEp-2 cells. The quantities of IFNs are of 5 IU/mL (0.5 ng/mL) of IFN gamma with 75 IU/mL (0.375 ng/mL) of IFN alpha 2b. With that optimum combination is reached a reduction the cell growth in vitro in a 76%.
Example 4: Effect of the pH, the ionic species and the concentration of the buffer solution in the stability of the mixture of the interferons alpha-2b and gamma in water solution.
[0059] To study the stability of the liquids and freeze-dried formulations of the synergistic compositions of the recombinant interferons gamma and alpha, the IFNs were diluted from their corresponding Active Pharmaceutical Ingredient (IFA) in different assay formulations: buffer solutions, buffer solutions mixture with individual excipients and buffer solutions mixture with various excipients. Representative samples of the vials of the different formulations were submitted to different treatment to evaluate the stability of the interferons: Cycles of freezing-thawing, lyophilization , agitation to 37°C, effect of the light and of the temperature. After the different processing, the physical-chemical stability was evaluated through different assays: physical appearance, sodium dodecilosulphate polyacrylamide gel electrophoresis (SDS-PAGE), reverse-phase liquid chromatography (RP-HPLC) and chromatography of molecular exclusion (ME-HPLC). The biological stability was evaluated by inmuno-enzymatic assays (ELISA) specific for each interferon and by biological assays of inhibition of the viral cytophatogenic effect. All these formulation design studies were carried out with the mixture of intermediate concentrations of the interferons (0.5 MIU of IFN gamma and 3.0 MIU of IFN alpha 2b). With the final variants of formulation obtained (prepared by duplicate) the synergistic compositions were prepared and its stability evaluated.
Organoleptic characteristic: It was determined by the analysis of the appearance of the formulation (if was maintained colorless, transparent and without aggregation of proteins). In the freeze-dried formulation, also the appearance of the freeze-dried product was analyzed.
The humidity: The content of residual humidity of the freeze-dried product was determined by the technique of Karl Fischer yodometric titration, employing a meter of humidity Radiometer (Model TIM 550).
Chemical stability: The purity of the proteins and the magnitude of the degradation was determined by RP-HPLC in a column C8 Vydac equipped with a keeping-column C8 Vydac (Vydac, Hesperia, CA) using a HPLC system Merck-Hitachi equipped with a system of solvent liberation, a diode arrangement detector, an oven and a data processing system. The purity of the proteins also was determined by SDS-PAGE.
Aggregates determination: The aggregation of the recombinant IFN gamma and of the IFN alpha 2b was measured by molecular exclusion HPLC using a Superdex-75 HR 10/30 column (Amersham Pharmacia Biotech AB, Sweden) and a HPLC system Merck-Hitachi equipped with a solvent liberation system, a diode arrangement detector, an oven and a data processing system. The content of covalent aggregates was determined by SDS-PAGE.
Alpha interferon ELISA: This assay has been developed in our laboratory (H. Santana., Espino Y., et al. (1999) A sandwich-type enzyme-linked immunosorbent assay for the analysis of recombinant human interferon α-2b. Biotechnology Techniques, 13, 341-346). The assay employs monoclonal antibodies and was carried out following the reported methodology. The measurement is reported here as percentage of the residual ELISA activity of the interferon alfa-2b in the different samples from each formulation variants, taking the ELISA activity in the initial sample as 100%.
Interferon gamma ELISA: This assay has been developed in our laboratory (Bouyón R., Santana H., et al. (2003) Development and validation of an enzyme-linked immunosorbent assay (ELISA) for recombinant human gamma interferon. Journal of Immunoassay and Immunochemistry, 24:1-10). The assay employs monoclonal antibodies and was carried out following the reported methodology. The measurement is reported here as percentage of the residual ELISA activity of the interferon gamma in the different samples from each formulation variants, taking the activity ELISA in the initial sample as 100%.
Antiviral biological activity quantification: The measurement of the biological activity was carried out as described Ferrero J., Ochagavia ME., et al. (1994, Titulación de la actividad antiviral del interferón utilizando el sistema de equipos SUMA. Biotecnología Aplicada, 11:34-42). The calculation of the biological activity was carried out as describes Ferrero J., Duany L., et al. (1997, Nuevo programa de cálculo, cuantificación de la actividad antiviral de interferones mediante la inhibición del efecto citopatogénico utilizando el sistema de equipos SUMA. Biotecnología Aplicada, 14: 267-269). The biological activity is reported here as percentage of the residual biological activity, taking the biological activity of the initial sample as 100%. To know the effect of the pH in the stability of the active ingredients, different formulations containing 0.5 MIU of IFN gamma and 3.0 MIU of IFN alpha 2b in different buffer solutions were prepared; that is to say, buffer citrate/phosphate, buffer phosphate, buffer citrate and buffer acetate. The formulations containing the mixtures of recombinant IFN gamma and of IFN alpha 2b with the buffer solutions and submitted to cycles of freezing-thawing or stored at 45°C and analyzed by ELISA at different time intervals. The formulations were prepared as to have the pH between 4 and 8, and all the buffer solutions at a concentration of 0.1 M. The tables 2, 3 and 4 reported the results of the assays carried out after 3 cycles of freezing-thawing and at different intervals of time, from 3 to 12 days at of 45°C.
Table 2. Stability at 45°C and during freezing-thawing cycles of recombinant IFN gamma
and IFN alpha 2b mixture (0.5 MIU IFN gamma and 3.0 MIU IFN alpha 2b) in water buffer
solution citrate/phosphate 0.1 M at different pH. Concentration of IFN gamma and IFN
alpha 2b in percentage (%).
| FREEZING-THAWING | INCUBATION AT 45°C | ||||||||
| ELISA IFNγ | ELISA IFNα | ELISA IFNγ | ELISA IFNα | ||||||
| T=0 | 3D | 6D | 12D | 3D | 6D | 12D | |||
| IFN/4 | 100 | 25.9 | 73.0 | 4.5 | ND | ND | 78.5 | 69.9 | 36.9 |
| 2690650 | IU/ML | ||||||||
| IFN/5 | 100 | 82.1 | 86.5 | 5.4 | ND | ND | 81.4 | 62.6 | 31.7 |
| 3820902 | IU/ML | ||||||||
| IFN/6 | 100 | 63.3 | 82.7 | 36.5 | 5.5 | 3.6 | 79.8 | 61.0 | 38.5 |
| 3532107 | IU/ML | ||||||||
| IFN/7 | 100 | 63.0 | 82.4 | 44.4 | 26.4 | 14.0 | 78.0 | 63.1 | 32.0 |
| 3532100 | IU/ML | ||||||||
| IFN/8 | 100 | 55.1 | 76.7 | 37.6 | 21.6 | 11.5 | 82.5 | 60.3 | 34.2 |
| 3179500 | IU/ML | ||||||||
| D = Days; ND = not determinable. IFN/4 = formulation in buffer citrate/phosphate pH 4.0; IFN/5 = formulation in buffer citrate/phosphate pH 5.0; IFN/6 = formulation in buffer citrate/phosphate pH 6.0; IFN/7 = formulation in buffer citrate/phosphate pH 7.0; IFN/8 = formulation in inkpad citrate/phosphate pH 8.0. |
Table 3. Stability at 45°C and during freezing-thawing cycles of recombinant IFN gamma
and IFN alpha 2b mixture (0.5 MIU IFN gamma and 3.0 MIU IFN alpha 2b) in water buffer
solution of phosphate 0.1 M at different pH. Concentration of IFN gamma and IFN alpha
2b in percentage (%).
| FREEZING-THAWING | INCUBATION AT 45°C | ||||||||
| ELISA IFNγ | ELISA IFNα | ELISA IFNγ | ELISA IFNα | ||||||
| T=0 | 3D | 6D | 12D | 3D | 6D | 12D | |||
| IFN/5 | 100 | 90.2 | 83.5 | 67.3 | 47.6 | 25.4 | 74.1 | 53.9 | 32,6 |
| 3549425 | IU/ML | ||||||||
| IFN/6 | 100 | 67.8 | 75.6 | 42.4 | 28.6 | 10.5 | 82.7 | 59.6 | 41.1 |
| 3459600 | IU/ML | ||||||||
| IFN/7 | 100 | 57.9 | 80.4 | 40.5 | 21.5 | 8.1 | 73.6 | 52.9 | 33.0 |
| 3523210 | IU/ML | ||||||||
| IFN/8 | 100 | 57.2 | 78.5 | 26.1 | 10.7 | 6.5 | 74.9 | 59.0 | 37.3 |
| 3321090 | IU/ML | ||||||||
| D = Days; ND = not determinable. IFN/5 = formulation in buffer phosphate pH 5.0; IFN/6 = formulation in buffer phosphate pH 6.0; IFN/7 = formulation in buffer phosphate pH 7.0; IFN/8 = formulation in buffer phosphate pH 8.0 |
[0060] The data from these tables (2,3,4) indicate that the formulations with values of pH between 5 and 8 have an adequate stability during the freezing-thawing cycles, preferably for the pH close to 5 and to 7 and in the buffer acetate, phosphate and citrate-phosphate. The thermal stability in water solution was greater to values of pH between 5 and 5.6, preferably in the buffer acetate and phosphate. The data from table 5 indicate that the formulations of higher buffer concentration showed better stability during the freezing-thawing cycles than those of lower concentration to pH 5.5, particularly for the IFN gamma in the different evaluated buffers. Nevertheless, the results of the thermal stability were better for pH 5.5 in the buffer acetate followed by phosphate. Dependence from the concentration was not observed. The thermal stability in buffer citrate-phosphate was better in buffer of low concentrations.
Table 4. Stability at 45°C and during freezing-thawing cycles of recombinant IFN gamma
and IFN alpha 2b mixture (0.5 MIU IFN gamma and 3.0 MIU IFN alpha 2b) in water buffer
solution of citrate and acetate 0.1 M at different pH. Concentration of IFN gamma
and IFN alpha 2b in percentage (%).
| FREEZING-THAWING | INCUBATION AT 45°C | ||||||||
| ELISA IFNγ | ELISA IFNα | ELISA IFNγ | ELISA IFNα | ||||||
| T=0 | 3D | 6D | 12D | 3D | 6D | 12D | |||
| IFN/C4 | 100 | 11.9 | 77.0 | 1.3 | ND | ND | 66.9 | 49.9 | 27.0 |
| 2690650 | IU/ML | ||||||||
| IFN/C5 | 100 | 64.6 | 78.1 | 2.7 | ND | ND | 71.3 | 56.6 | 41.8 |
| 3820902 | IU/ML | ||||||||
| IFN/C6 | 100 | 65.8 | 66.9 | 12.0 | 5.9 | ND | 61.5 | 43.7 | 28.2 |
| 3532101 | IU/ML | ||||||||
| IFN/A4 | 100 | 87.1 | 82.3 | 65.9 | 46.4 | 27.9 | 64.5 | 46.6 | 29.0 |
| 3532100 | IU/ML | ||||||||
| IFN/A5 | 100 | 86.1 | 79.7 | 59.4 | 45.9 | 24.0 | 68.3 | 47.9 | 30.7 |
| 3179500 | IU/ML | ||||||||
| IFN/A5.6 | 100 | 74.8 | 75.0 | 50.8 | 31.3 | 18.9 | 67.7 | 48.3 | 27.6 |
| 3179500 | IU/ML | ||||||||
| D = Days; ND = not determinable; IFN/C4 = formulation in buffer citrate pH 4.0; IFN/C5 = formulation in buffer citrate pH 5.0; IFN/C6 = formulation in buffer citrate H 6.0; IFN/A4 = formulation in buffer acetate pH 4.0; IFN/A5 = formulation in buffer acetate pH 5.0; IFN/A5.6 = formulation in buffer acetate pH 5.6. |
Table 5. Stability at 45°C and during freezing-thawing cycles of recombinant IFN gamma
and IFN alpha 2b mixture (0.5 MIU IFN gamma and 3.0 MIU IFN alpha 2b) in water solution
pH 5.5 in buffer citrate-phosphate, phosphate and acetate at different concentrations.
Concentration of IFN gamma and IFN alpha 2b in percentage (%).
| FREEZING-THAWING | INCUBATION AT 45°C | ||||||||
| ELISA IFNγ | ELISA IFNα | ELISA IFNγ | ELISA IFNα | ||||||
| T=0 | 3D | 6D | 12D | 3D | 6D | 12D | |||
| IFN/C-F25 | 100 | 59.2 | 77.2 | 62.8 | 21.7 | 27.9 | 67.2 | 59.9 | 38.7 |
| 2690650 | IU/ML | ||||||||
| IFN/C-F50 | 100 | 61.9 | 79.6 | 45.5 | 26.4 | 16.6 | 71.9 | 62.3 | 36.1 |
| 3820902 | IU/ML | ||||||||
| IFN/C-F100 | 100 | 63.3 | 82.7 | 36.5 | 5.5 | 3.6 | 79.8 | 61.0 | 38.5 |
| 3532107 | IU/ML | ||||||||
| IFN/Fk25 | 100 | 70.9 | 73.0 | 71.2 | 50.9 | 30.5 | 61.4 | 54.4 | 36.8 |
| 3532100 | IU/ML | ||||||||
| IFN/Fk50 | 100 | 79.7 | 75.9 | 69.1 | 48.7 | 28.7 | 63.6 | 52.9 | 31.6 |
| 3179500 | IU/ML | ||||||||
| IFN/Fk100 | 100 | 90.2 | 83.5 | 67.3 | 47.6 | 25.4 | 74.1 | 53.9 | 32.6 |
| 3179500 | IU/ML | ||||||||
| IFN/FNa | 100 | 70.3 | 77.6 | 68.4 | 47.3 | 24.3 | 63.6 | 56.1 | 37.7 |
| 3532100 | IU/ML | ||||||||
| IFN/FNa 50 | 100 | 82.8 | 80.1 | 60.8 | 46.5 | 21.4 | 71.3 | 53.1 | 34.6 |
| 3179500 | IU/ML | ||||||||
| IFN/FNa100 | 100 | 87.9 | 86.5 | 73.9 | 42.0 | 32.5 | 74.7 | 52.9 | 35:0 |
| 3179500 | IU/ML | ||||||||
| IFN/A25 | 100 | 62.6 | 66.3 | 73.4 | 45.3 | 34.2 | 63.1 | 44.3 | 28.4 |
| 3532100 | IU/ML | ||||||||
| IFN/A50 | 100 | 69.3 | 69.0 | 67.3 | 42.9 | 29.7 | 65.6 | 51.9 | 33.2 |
| 3179500 | IU/ML | ||||||||
| IFN/A100 | 100 | 76.4 | 77.1 | 52.6 | 30.1 | 22.8 | 70.5 | 49.0 | 26.8 |
| 3179500 | IU/ML | ||||||||
| D: Days; ND: not determinable; IFN/C-F25: formulation in buffer citrate-phosphate pH 5.5, 25 mM; IFN/C-F50: formulation in buffer citrate-phosphate pH 5.5, 50 mM; IFN/C-F100: formulation in buffer citrate-phosphate pH 5.5, 100 mM; IFN/Fk25: formulation in buffer potassium-phosphate pH 5.5, 25 mM; IFN/Fk50: formulation in buffer potassium-phosphate pH 5.5, 50 mM; IFN/Fk100: formulation in buffer phosphate 5.5, 100 mM; IFN/FNA 25: formulation in buffer sodium phosphate pH 5.5, 25 mM; IFN/FNA 50: formulation in buffer sodium phosphate pH 5.5, 50 mM; IFN/FNA 100: formulation in buffer sodium phosphate pH 5.5, 100 mM; IFN/A 25: formulation in buffer acetate pH 5.5, 25 mM; IFN/A 50: formulation in buffer acetate pH 5.5, 50 mM; IFN/A 100: formulation in buffer acetate pH 5.5,100 mM. |
Example 5. Freeze-dried formulation (1.4 x 106 IU of IFN gamma and 1.7 x 106 IU of IFN alpha 2b per vial).
[0061] Composition: IFN gamma 2.8 x 108 IU, IFN alpha 2b 3.4 x 108 IU, di-hydrogen potassium phosphate 0.0802 g, di-sodium di-hydrated hydrogen phosphate 0.249 g, saccharose 4 g, glycine 0.8 g, Tween 20 0.03 g, polyetilenglycol 6000 1 g, water for injection sufficient quantity for 100 mL.
All the components except the interferons were measured and diluted with water for injection. The pH of the solution is checked and if is necessary, adjusted to a value of 7.2± 0.2 with diluted (1:2) acetic acid or with 1 M of NaOH. The active pharmaceutical ingredients of IFN gamma and IFN alpha 2b were added and diluted to the appropriate concentration. The solution was filtered in sterile form and the vials filled and caped with plugs for the lyophilization in a class 100 area, where the process was carried out. Finally, the vials are covered and sealed; and the product stored between 2 and 8°C. The table 6 shows the main parameters of the lyophilization cycle employed.
Table 6. Summary of the parameters of the lyophilization cycle. Chronogram of temperature
per phases.
| Cycle steps | Temperature | Duration |
| Freezing | -45 °C | 2 hours |
| -20 °C | 2 hours | |
| -45 °C | 6 hours | |
| Primary driying | -35 °C | 12 hours |
| Secundary driying | 25°C | 12 hours |
[0062] At established intervals of time samples were taken and analyzed the content of residual humidity of the product, the content of IFN gamma and IFN alpha 2b (by ELISA), the biological activity, the purity by RP-HPLC and the appearance of the freeze-dried product as well as it reconstituted. The results are presented in the table 7.
Table 7. Data of residual humidity of the product, content of IFN gamma and IFN alpha
2b, the biological activity, purity by RP-HPLC. Temperature 5°C. FREEZE-DRIED FORMULATION:
1.4 MIU IFN gamma and 1.7 MIU IFN alpha 2b/vial.
| Time (month) | Residual Humidity (%) | ELISA | Antiviral Activity | Purity RP-HPLC | Description | |||
| IFNγ (µg/vial*) | IFNα (µg/vial*) | IU/vial* | Total (%) | IFNγ (%) | IFNα (%) | |||
| Inicial | 1.4 | 315.8 | 21.9 | 2.51 | 97.8 | 90.6 | 7.2 | STI |
| 1 | - | 291.2 | 24.7 | 3.78 | 96.4 | 87.7 | 8.7 | STI |
| 3 | - | 307.5 | 23.5 | 3.17 | 97.7 | 87.9 | 9.8 | STI |
| 6 | 2.6 | 279.1 | 22.6 | 2.89 | 97.0 | 88.9 | 8.2 | STI |
| *The filled volume was 0.5 mL/vial; STI: Uniform white lyophilized; after reconstitution, a transparent colorless solution, essentially free of particles. |
Example 6. Freeze-dried formulation (0.5 x 106 IU of IFN gamma and 3.0 x 106 IU of IFN alpha 2b per vial).
[0063] Composition: 1.0 x 108 UI, IFN alpha 2b 6.0 x 108 UI, di-hydrogen potassium phosphate 0.0802 g, di-sodium di-hydrated hydrogen phosphate 0.249 g, saccharose 4 g, glycine 0.8 g, Tween 20 0.03 g, polyetilenglycol 6000 1 g, water for injection sufficient quantity for 100 mL. The method of preparation was the same described in the freeze-dried formulation of the example 5.
[0064] At established intervals of time samples were taken and analyzed the content of residual humidity of the product, the content of IFN gamma and IFN alpha 2b (by ELISA), the biological activity, the purity by RP-HPLC and the appearance of the freeze-dried product as well as it reconstituted. The results are presented in the table 8.
Table 8. Data of residual humidity of the product, content of IFN gamma and IFN alpha
2b, the biological activity, purity by RP-HPLC. Temperature 5°C. FREEZE-DRIED FORMULATION:
0.5 MIU IFN gamma and 3.0 MIU IFN alpha 2b/vial.
| Time (month) | Residual humidity (%) | ELISA | Antiviral Activity | Purity RP-HPLC | Description | |||
| IFNγ (µg/vial*) | IFNα (µg/vial*) | IU/vial* | total % | IFNα (%) | IFNγ (%) | |||
| Inicial | 1.3 | 93.7 | 38.5 | 3.15 | 97.5 | 28.1 | 69.4 | STI |
| 1 | - | 104.5 | 43.9 | 4.41 | 96.8 | 29.5 | 67.3 | STI |
| 3 | - | 98.5 | 36.4 | 2.69 | 97.1 | 27.6 | 69.5 | STI |
| 6 | 1.9 | 108.2 | 44.0 | 3.91 | 96.9 | 28.8 | 68.1 | STI |
| *The filled volume was 0.5 mL/vial; STI: Uniform white lyophilized; after reconstitution, a transparent colorless solution, essentially free of particles. |
Example 7: Clinical trial with the stabilized freeze-dried pharmaceutical formulation (0.5 x 106 IU of IFN gamma and 3.0 x 106 IU of IFN alpha 2b per vial). Application intralesional in the CBC.
[0065] The stable freeze-dried pharmaceutical formulation described in the example 6 was employed in the execution of a triple blind, controlled, randomized clinical trial, that included 59 patients with clinical and histological diagnosis of CBC of any location and type of skin with lesions of a diameter less than four centimeters. The patients were assigned to three groups of treatment by randomization. The lesions were treated intralesional with the half of the doses of recombinant IFN alpha 2b (1,5 x106 IU/mL); or recombinant IFN gamma (0, 25 x106 IU/mL) or the stable freeze-dried formulation (0.5 x 106 IU of IFN gamma and 3.0 x 106 IU of IFN alpha 2b per vial), group I, II and III, respectively. The IFN were applied three times per weeks, during three consecutive weeks, continuing during 9 weeks, once a week, or until the total disappearance of the lesion, moment in which the clinical efficacy of the treatment was evaluated. The 9.5%, 35,3% and 5,3% of the lesions of the group with IFN alpha 2b, IFN gamma and the formulation (0.5 x 106 IU of IFN gamma and 3.0 x 106 IU of IFN alpha 2b per vial), respectively diminished in less than 50% the size of the lesion. In the remainder lesions a 90,5%, 57.9% and 94,7% (group I, II and III, respectively) of objective response (total disappearance or decrease of more than the 50% of the initial size) was observed. None of the lesions progressed (to see table 9). In the stratum of the "patient that did not finish the treatment", a superiority of the treatment with the formulation that contains the recombinant IFNs gamma and alpha 2b with synergistic antiproliferative effect is observed (17% of difference with respect to the treatment with IFN alpha 2b and 27% of difference with respect to IFN gamma). In the stratum of the "patient with less than 11 weeks of treatment", a superiority of the formulation is observed. Approximately 30% of difference with respect to IFN gamma and 27% with respect to IFN alpha 2b. The proportion of complete response is higher (> 40% of superiority with respect to IFN alpha 2b and >30% with respect to IFN gamma treatment). Patients with less than 12 injections, a 100% of favorable response were achieved (of them 50% of RC) in the group of the therapy with the formulation. In the other 2 treatment groups, the percentage of favorable response was of 67% (of them the 33.3% of RC), that is to say is achieved approximately a 33% of difference in favor of the combined therapy. See table 9.
Table 9. Evaluation of the clinical response stratifying according to time of treatment
with the half dose FREEZE-DRIED FORMULATION: 0.5 MIU IFN gamma and 3.0 MIU IFN alpha
2b/vial.
| Variables | IFN alfa | IFN gamma | Formulación | P (Fisher) | ||||
| Terminaron tratamiento | Completa | 7(41.2%) | 17 (100%) | 4(33.3%) | 9 (75.0%) | 8 (50%) | 16 (100%) | I - II p= 0.060 III - II p= 0.067 |
| Parcial | 10 (58.8%) | 5 (41.7%) | 8 (50%) | |||||
| Estable | 0(0%) | 3(25.0%) | 0(0%) | |||||
| No | Completa | 0(0%) | 2 (50%) | 0 (0%) | 2 (40%) | 0 (0%) | 2 (66.7%) | I - II p= 1.000 |
| terminaron tratamiento | Parcial | 2 (50%) | 2 (28.6%) | 2 (66.7%) | III -II p= 1.000 | |||
| Estable | 2 (50%) | 3(60%) | 1(33.3%) | |||||
| Tiempo de tratamiento >= 11 semanas | Completa | 6 (37.5%) | 16 (100%) | 2 (20.0%) | 7 (70%) | 3 (27.3%) | 11 (100%) | I - II p= 0.046* III-II p= 0.090 |
| Parcial | 10(62.5%) | 5(50.0%) | 8 (72.7%) | |||||
| Estable | 0(0%) | 3 (30%) | 0 (0%) | |||||
| Tiempo de tratamiento < 11 semanas | Completa | 1(20%) | 3 (60%) | 2 (28.6%) | 4 (57.1%) | 5 (62.5%) | 7(87.5%) | I- II p= 0.689 III - II p= 0.230 |
| Parcial | 2(40%) | 2 (28.6%) | 2 (25.0%) | |||||
| Estable | 2(40%) | 3 (42.9%) | 1(12.5%) | |||||
| < 12 Inyecciones recibidas | Completa | 1 (33.3%) | 2 (66.7%) | 2 (33.3%) | 4(66.7%) | 3 (50%) | 6 (100%) | I -II p= 1.000 III-II p= 0.455 |
| Parcial | 1 (33.3%) | 2(33.3%) | 3(50%) | |||||
| Estable | 1 (33.3%) | 2 (33.3%) | 0 (0%) | |||||
| >12 Inyecciones recibidas | Completa | 6 (33.3%) | 17(94.4%) | 2 (18.2%) | 7 (63.6%) | 5 (38.5%) | 12 (92.3%) | I- II p= 0.054 III - II p= 0.142 |
| Parcial | 11(61.1%) | 5 (45.4%) | 7 (53.8%) | |||||
| Estable | 1 (5.6%) | 4 (36.4%) | 1 (7.7%) | |||||
[0066] As we can observe in the table 10, with the stable freeze-dried formulation that contains the recombinant IFNs gamma and alpha 2b, it is possible to obtain clinical complete response in shorted period of time than with the interferons by separated, with a difference of approximately 4 weeks before with respect to IFN alpha.
Table 10. Evaluation of the time to the complete clinical response. Treatment with
the half dose freeze-dried formulation: 0.5 MU IFN gamma and 3.0 MIU IFN alpha 2blvial.
| Time to RC (Weeks) | IFN gamma (N = 17) | IFN alpha (N=21) | Formulation (N=19) | P (Kruskal-Wallis) |
| Median ± RQ | 9:0 ± 7.5 | 12.0 ± 1.0 | 8.5 ± 5.8 | 0.507 |
[0067] In no treated case the formation of keloid was observed, on the contrary all the treated cases had a good scar formation of the lesion with normal sensibility, normal elasticity or slightly diminished and absence of dryness, fragility and harshness. With respect to the color, in the majority of the patients treated with the formulation was observed a normal coloring in the treated place (47.4%), the double of observed for the group with IFN alpha (28.6%). At the end of trial, in the group treated with the formulation a greater percentage of flat wounds was observed (63.2%) with respect to the group treated with IFN alpha alone 2b (52.4%) which is shown in the table 11.
Table 11. Esthetics evaluation at the end of treatment. Treatment with the half dose
freeze-dried formulation: 0.5 MIU IFN gamma and 3.0 MIU IFN alpha 2b/vial.
| Variables | FN gamma (N=17) | IFN alpha (N=21)) | Formulation (N=19) | |
| Color | Normochromy | 8 (47.1 %) | 6 (28.6%) | 9 (47.4%) |
| Slightly hypochromy | 1 (5.9%) | 4 (19%) | 6 (31.6%) | |
| Hypochromy | 1 (5.9%) | 2 (9.5%) | 1 (5.3%) | |
| Slightly hyperchromy | 7(41.2%) | 8 (38.1%) | 3 (1.8%) | |
| Hyperchromy | - | 1 (4.8%) | - | |
| Volumen | Flat lesion | 12(70.6%) | 11 (52.4%) | 12(63.2%) |
| Slightly hypertrophy | 5(29.4%) | 10(47.6%) | 7(36.8%) | |
[0068] The combination of interferons did not potentate the adverse events, because there were not detected statistical differences between treatment groups, with respect to the production or intensity of them. In general, they were light (71.2%) or moderates and well tolerated. Themselves not adverse serious neither very serious events were presented. (Table 12).
[0069] As it can be observed in this table, the most frequent adverse events in each group of treatment were: fever (38.5%; 60.8% and 26.2%), myalgias (38.5%; 3.9% and 31%) and chills (12.8%; 19.6% and 21.4%) with IFN alpha, IFN gamma and the combination, respectively. The total of adverse events presented was slightly superior in the group of patients treated with IFN gamma. In general, the combined treatment achieved a 32% of superiority of complete response, approximately 4 weeks before and with less than 25% injections with respect to the IFN alpha group. The combination did not promote adverse events and any recurrence was found during the follow-up one year after finalized the treatment for patients with complete clinical response. Since the cosmetic point of view, the result was very good resulting mostly in flat and normochromics wounds.
Example 8: Results of the compasional use of the mixture of recombinant IFNs gamma and alpha 2b in patients with skin tumors, not susceptible of standard treatment. Cases report.
Patient 1
[0070] Patient EPR: HC: 302396 Age: 82 years, Sex: Male, personal pathological antecedent (APP): n/r Remitted to National Institute of Oncology and Radiology (INOR) 17/10/01 with tumor of the skin in the anterior region of thorax, had received electrofulguration treatment that after this grew in ulcerous form, having been surgically excision the 03/07/01. Result: Spinocellular carcinoma incompletely excised. The patient arrives with persistent tumoral lesion of 3 cm in the place of original tumor, with high edges. At physical observation did not present regional metastasic ganglion. Radiation therapy on the tumor was indicated, and ended it the 29/01/02: 60co 50 Gy + X-Rays 12 Gy. Total dose per tumor was 62gy. A month later, the patient showed fixed tumor to the collarbone and at lower third of the esternocleidomastoide muscle. In short time, the tumor continued a fast growth, showing the 04/03/02 great-ulcerated wound of 10 x 8 cm on the internal third of the right collarbone, base of the neck and part of the sternum, all toward the previous part of the thorax. A surgical intervention was proposed to him. It was refused to be done a surgical intervention, because of his 82 years of age and to have high surgical risk. Then, a treatment with intra-lesional IFN was recommended.
The tumor with high size (12.5 x 9 cm and 1-1.5 cm in thickness), was fixed to bone and muscle. The IFN application was planned in three sectors of perimeter. Each sector was infiltrated with 1.5 mL of solution in approximately 5 cm3 of tissue (1.5 x 1.5 x 1.5) to a dose of 0.5 x 106 IU of IFN gamma + 6 x 106 IU of IFN alpha 2b in 6 mL of water for injection, three times per week for three weeks (figure 7a). After the fifth application of the product (second week) is decided to applied a higher dose of IFN gamma (double, 1 x 106 IU). The dose escalation was proposed trying to obtain a better result of a so large tumor, because of the absence of adverse effects in the previous dosage, and on the prior information of the clinical study where the synergistic effect of both interferons was shown.
Overall, the patient received 27 applications for a total dose of 25 x 106 IU of IFN gamma + 162 x 106 IU of IFN alpha 2b in two months of treatment. In the fifth application, it was appreciated a leveling of the edge of the wound to the level of the normal skin, (figure 7b). A month after beginning the treatment, the patient refers intense pain of the right upper member being observed infiltration of the brachial nervous plexus, exposition, necrosis and fracture of the collarbone. At application #20 was observed that in the places injected stopped the tumoral growth, but not thus in the center of the tumor where grew in lobular-like shape, (figure 7c). This condition was observed even at the application 24. Besides appeared sepsis and necrosis in the center of the tumoral ulcer. Exactly two months after started the treatment (application #27) the patient suffered intense arterial bleeding by infiltration of the artery subclavia; the hemoglobin of the patient lowered to 80 g/L, and the treatment was interrupted for 15 days, at the end of which returned due to a continuing bleeding and to have progressive asthenia. The patient died two months later by arterial break. The places injected were maintained without tumor growth. In the first eight applications, some adverse events were registered like fever (39°C), chills, perilesional erythema and asthenia, but all of slightly intensities and short term. Conclusions: Clinical response in the places injected was reached, lasting by at least two months, adverse effects were without importance.
Patient 2
[0071] Patient LGR: HC: 158390 Age: 65 years Sex: Female, APP: n/r. Patient that suffers of multiple carcinomas based on all the face. The patient was intervened surgically and radiated several times in the lower eyelid of the left eye with grafts. Now shows tumor recurrence of 5mm of diameter in the edge of the eyelid and another flat scar-like wound under the eyelid toward the cheekbone, (figure 8a). The alternative of treatment would be a new surgery with the objections to be a case already multi-treated. In the upper eyelid of the same eye has another basal carcinoma of 7 mm that has not been previously treated.
08/05/02 is proposed the treatment with intralesional IFN (0.5 x 106 IU of IFN gamma + 3 x 106 IU of IFN alpha 2b) in 4 mL, three times for week for three weeks. Overall, the patient received 10 applications for a total dose of 35 x 106 IU of interferon (5 x 106 IU of IFN gamma + 30 x 106 IU of IFN alpha 2b). In the quarter infiltration, the scar-like wound disappeared and in the eyelid had an ulcer necrotized in the place of the tumor, (figure 8b). Two months after the treatment, not tumor in the eyelid and disappearance of the scar-like flat wound of the cheekbone is observed (figure 8c).
A local-regional effect was obtained. It was observed a reduction in 50% of the basal carcinoma of the upper eyelid of the left eye that was not treated directly with IFN that then was surgical excised. Three years later the patient remains still controlled of the infiltrated lesions with IFN. Some adverse events of light intensities and of short term were registered like fever (39°C), chills and chemosis in the treated eye that was alleviated with cold compresses.
Conclusions: Patient with complete clinical response until August of the 2005 (last control), minimum adverse effects.
Example 9. Freeze-dried formulation (0.5 x 106 IU of IFN gamma and 10 x 106 IU of IFN alpha 2b per vial).
[0072] Composition: IFN gamma 1.0 x 108 IU, IFN alpha 2b 20 x 108 IU, potassium di-hydrogen phosphate 0.0802 g, di-sodium di-hydrated hydrogen phosphate 0.249 g, saccharose 4 g, glycine 0.8 g, Tween 20 0.03 g, polyetilenglycol 6000 1 g, water for injection sufficient quantity for 100 mL. The method of preparation was the same that was described in the freeze-dried formulation of the example 5. At established intervals of time samples were taken and analyzed the content of residual humidity of the product, the content of IFN gamma and IFN alpha 2b (by ELISA), the biological activity, the purity by RP-HPLC and the appearance of the freeze-dried product as well as it reconstituted. The results are presented in the table 13.
Table 13. Data of residual humidity of the product, content of IFN gamma and IFN alpha
2b, the biological activity, and purity by RP-HPLC. Temperature 5°C. FREEZE-DRIED
FORMULATION: 0.5 MIU IFN gamma and 10.0 MIU IFN alpha 2b/vial.
| Time (month) | Residual Humidity % | ELISA | Activity | Purity RP-HPLC | Description | |||
| IFNγ (µg/vial*) | IFNα (µg/vial*) | Antiviral (IU/vial*) | Total (%) | IFNγ (%) | IFNα (%) | |||
| Inicial | 0.9 | 94.0 | 142.2 | 12.90 | 98.8 | 43.7 | 55.1 | STI |
| 1 | - | 105.5 | 133.2 | 8.37 | 97.7 | 40.8 | 56.9 | STI |
| 3 | - | 102.1 | 129.4 | 11.55 | 97.6 | 43.0 | 54.5 | STI |
| 6 | 1.7 | 91.7 | 137.1 | 9.13 | 95.4 | 39.4 | 57.0 | STI |
| *The filled volume was 0.5 mL/vial; STI: Uniform white lyophilized; after reconstitution, a transparent colorless solution, essentially free of particles. |
Example 10. Employment of stable freeze-dried formulation composed by 0.5 MIU IFN gamma and 10.0 MIU IFN alpha 2b/vial in combination with Cisplatin. Report of case.
Patient 3
[0073] Patient JGA: HC: Age: 33 years, Sex: Male, APP: Patient n/r recorded in the INOR with a carcinoma of basal cells that penetrates the internal angle of the left eye, with several surgical interventions, and radiated. Now, the patient has a tumor ulcerated that arrives to the bones of the base of the skull (figure 9a). Verified by axial computerized tomography (TAC) is observed cavity in the own bones of the nose and of the internal wall of the orbit, unbearable fetidness that leaves for the left nostril and purulent yellow secretion at the same place.
Due to the extension of the wound was decided to do a combined treatment with systemic chemotherapy with cysplatin to dose of 6 cycles with intervals of 21 days and at same time the formulation (0.5 MIU IFN gamma and 10.0 MIU IFN alpha 2b/vial) infiltrated locally 3 times for week for three weeks.
At the end of the third application, already important partial clinical response was observed, that permit the palpebral opening and the decrease of the fetidness. It was present at the same time chemosis of moderate intensity. A complete clinical response is appreciated after a month. This response is maintained until the end of chemotherapy. The adverse events were few, something fever and chills and pain referred in the place of the scar of the wound. A year later the patient maintains the complete clinical response (figure 9b).
Example 11. Liquid stable pharmaceutical formulation (1.4 x 106 IU of IFN gamma and 1.7 x 106 IU of IFN alpha 2b per vial).
[0074] Composition: IFN gamma 2.8 x 108 IU, IFN alpha 2b 3.4 x 108 IU, sodium acetate 0.708 g, acetic acid 0.079 mL, Tween 20 0.01 g, manitol 5 g, water for injection sufficient quantity for 100 mL.
All the components except the interferons were measured and suspended with water for injection. The pH of the solution was checked and, if is necessary, adjusted to value of 5.5± 0.2 with acetic acid diluted (1:2) or with 1 M of NaOH. The active pharmaceutical ingredients of recombinant IFN gamma and IFN alpha 2b were added and diluted to the appropriate concentration.
The solution was filtered in sterile condition. The vials were filled with the formulation and covered and sealed in a class 100 area. Finally, the product is stored between 2 and 8°C. Several samples were taken from the manufactured formulation, and stored at 8°C and 2°C for a period of six months.
At established intervals of time samples were taken and analyzed the content of residual humidity of the product, the content of IFN gamma and IFN alpha 2b (by ELISA), the biological activity, the purity by RP-HPLC and the appearance of the freeze-dried product as well as it reconstituted. The results are presented in the table 14.
Table 14. Data of pH of the product, content of IFN gamma and IFN alpha 2b, the biological
activity, and purity by RP-HPLC. Temperature 5°C. LIQUID FORMULATION: 1.4 MIU IFN
gamma and 1.7 MIU IFN alpha 2b/vial.
| Time (months) | pH | ELISA | Activity | Purity, RP-HPLC | Description | |||
| IFNγ (µg/vial*) | IFNα (µg/vial*) | Antiviral IU/vial* | Total (%) | IFNγ (%) | IFNα (%) | |||
| Inicial | 5.58 | 3052 | 24.9 | 3.66 | 96.9 | 88.1 | 8.8 | STI |
| 1 | 5.43 | 279.1 | 21.2 | 3.21 | 97.2 | 87.9 | 9.3 | STI |
| 3 | 5.56 | 285.4 | 20.7 | 2.47 | 95.8 | 89.7 | 7.1 | STI |
| 6 | 5.45 | 293.0 | 23.9 | 3.39 | 95.5 | 88.1 | 8.4 | STI |
| *The filled volume was 0.5 mL/vial; STI: Uniform white lyophilized; after reconstitution, a transparent colorless solution, essentially free of particles. |
Example 12. Liquid stable pharmaceutical formulation (0.5 x 106 IU of IFN gamma and 3.0 x 106 IU of IFN alpha 2b per vial).
[0075] Composition: IFN gamma 2.0 x 108 IU, IFN alfa 2b 12.0 x 108IU, sodium acetate 0.708 g, acetic acid 0.079 mL, Tween 20 0.01 g, manitol 5 g, water for injection sufficient quantity for 100 mL.
The method of preparation was the same that was described in the freeze-dried formulation of the example 11. At established intervals of time samples were taken and analyzed the content of residual humidity of the product, the content of IFN gamma and IFN alpha 2b (by ELISA), the biological activity, the purity by RP-HPLC and the appearance of the freeze-dried product as well as it reconstituted. The results are presented in the table 15.
Table 15. Data of pH of the product, content of IFN gamma and IFN alpha 2b, the biological
activity, and purity by RP-HPLC. Temperature 5°C. LIQUID FORMULATION: 0.5 MUI IFN
gamma and 3.0 MIU IFN alpha 2b/vial.
| Time (months) | pH | ELISA | Activity | Purity RP-HPLC | Description | |||
| IFNγ (µg/vial) | IFNα (µg/vial*) | Antiviral (IUvial*) | Total (%) | IFNγ (%) | IFNα (%) | |||
| Initial | 5.55 | 109.4 | 38.2 | 4.25 | 97.6 | 58.3 | 29.3 | STI |
| 1 | 5.37 | 103.7 | 39.5 | 2.73 | 97.1 | 59.8 | 27.3 | STI |
| 3 | 5.49 | 95.0 | 42.9 | 3.35 | 97.3 | 70.2 | 27.1 | STI |
| 6 | 5.58 | 94.9 | 36.2 | 3.91 | 96.8 | 58.5 | 28.3 | STI |
| *The filled volume was 0.5 mL/vial; STI: Uniform white lyophilized; after reconstitution, a transparent colorless solution, essentially free of particles. |
Example 13. Liquid stable pharmaceutical formulation (0.5 x 106 IU de IFN gamma and 10 x 106 IU de IFN alpha 2b per vial).
[0076] Composition: IFN gamma 2.0 x 108 IU, IFN alpha 2b 40 x 108 IU, sodium acetate 0.708 g, acetic acid 0.079 mL, Tween 20 0.01 g, manitol 5 g, water for injection sufficient quantity for 100 mL.
The method of preparation was the same that was described in the freeze-dried formulation of the example 11.
At established intervals of time samples were taken and analyzed the content of residual humidity of the product, the content of IFN gamma and IFN alpha 2b (by ELISA), the biological activity, the purity by RP-HPLC and the appearance of the freeze-dried product as well as it reconstituted. The results are presented in the table 16.
Table 16. Data of pH of the product, content of IFN gamma and IFN alpha 2b, the biological
activity, and purity by RP-HPLC. Temperature 5°C. LIQUID FORMULATION: 0.5 MIU IFN
gamma and 10.0 MIU IFN alpha 2b/vial.
| Time (months) | pH | ELISA | Activity | Purity RP-HPLC | Description | |||
| IFNγ (µg/vial*) | IFNα (µg/vial*) | Antiviral (IU/vial*) | Total (%) | IFNγ (%) | IFNα (%) | |||
| Initial | 5.53 | 91.9 | 138.2 | 12.41 | 98.0 | 39.1 | 58.9 | STI |
| 1 | 5.57 | 93.7 | 121.5 | 8.73 | 97.5 | 41.7 | 55.9 | STI |
| 3 | 5.49 | 105.0 | 142.9 | 9.46 | 95.8 | 38.2 | 58.6 | STI |
| 6 | 5.51 | 99.8 | 134.7 | 10.93 | 97.5 | 42.5 | 42.5 | STI |
| *The filled volume was 0.5 mL/vial; STI: Uniform white lyophilized; after reconstitution, a transparent colorless solution, essentially free of particles. |
Example 14. Semisolid pharmaceutical formulation (0.16 x 106 IU of IFN gamma and 1.0 x 106 IU of IFN alpha 2b per gram of semisolid).
[0077] Pharmaceutical formulation for topical application, preferably as a cream, unguent or gel. The pharmaceutical preparation contains recombinant interferon gamma and alpha interferon 2 as active principle. The composition is of 1.6 x107IU of IFN gamma and of 1 x 108 IU of IFN alpha 2b, sufficient quantity for 100 grams of semisolid.
Preparation of cream: To prepare the cream melts the solid petroleum jelly and the cetyl alcohol at 75°C and mixed with constant agitation, maintained to the end of the process. Once homogenized Tween 60 is incorporated to the mixture. On the other hand, the methyl- and propyl-paraben is dissolved in water at 90°C and is incorporated to the previous mixture when the temperature has reduced until 75°C. Subsequently the emulsion is cooling slowly until 37°C and is incorporated the water solution that contains the recombinant IFN gamma and IFN alpha 2b.The resultant cream is stored to 4°C in 15g tubes (to see table 17).
Table 17. Ingredients of the cream formulation.
| Ingredients | % |
| IFNγ | 2.2 |
| IFNα 2b | 0.58 |
| Cetyl Alcohol | 4 |
| Petroleum jelly | 10 |
| Tween 60 | 2 |
| Methyl-, propyl-paraben | 0,2 |
| Distilled water c.s.p | 81.2 |
[0078] Preparation of unguent: In a container, the parabens are dissolved in the water at 90 °C and then left to chill to 37°C. In another container the liquid petrolatum and the Span 20 are mixed with constant agitation. Subsequently, the content of both containers is mixed and when the temperature have diminished under 37°C is incorporated the recombinant IFN gamma and IFN alpha 2b, maintaining the constant agitation. Then it is incorporated the white petrolatum until achieving a homogenization. The resultant unguent is stored to 4° C in 15 g tubes (to see table 18).
Table 18. Ingredients of the unguent formulation.
| Ingredients | % |
| IFNγ | 2.2 |
| IFNα 2b | 0.58 |
| White solid petrolatum | 60 |
| Heavy liquid petrolatum | 10 |
| Span 20 | 3 |
| Methyl- propyl-paraben | 0.2 |
| Distilled water c.s.p | 24.02 |
[0079] Preparation of gel: The EDTA, parabens and alcohol are dissolved in separated containers and then is added the propylenglycol. Then these solutions with constant agitation are mixed and is incorporated slowly the carbopol 940 with vigorous agitation until obtaining a murky dispersion without presence of lumps. It is prepared separated, in an adequate container, a 1 N sodium hydroxide solution and is added slowly with agitation to the dispersion that contains the remainder of the components of the formulation. Subsequently the IFN gamma and IFN alpha 2b are incorporated with gentle agitation. Once the gel is formed is bottled in 15 g tubes at 4°C (to see table 19).
Table 19. Ingredients of the gel formulation.
| Ingredients | % |
| IFNγ | 2.2 |
| IFNα 2b | 0.58 |
| Carbopol 940 | 0.5 |
| Propylene glycol | 10 |
| Methylparaben and Propylparaben | 0.2 |
| Sodium hydroxide | 0.2 |
| Calcium disodium ethylene diamine tetra-acetate (EDTA) | 0.01 |
| Ethanol | 2 |
| Distilled water c.s.p | 84.31 |
1. Stablilized pharmaceutical formulations for parental (liquid or freeze-dried), or
topical (gel, unguent, cream) administration, that comprise different quantities of
recombinant interferons gamma and alpha in synergistic proportions for the treatment
of pathological events characterized by benign non-physiological or malignant cellular growth of tissues or organs and that
comprise additionally, pharmaceutically acceptable excipients or carriers.
2. Freeze-dried stablilized pharmaceutical formulations as described in claim 1, which
comprise a buffer solution and at least a component selected from non-reducing sugar
compounds, amino acids, surfactants and stabilizing polymers.
3. Freeze-dried stablilized pharmaceutical formulations as described in claim 2, where
is employed a buffer with the capacity to maintain the pH between 4.9 and 7.5, as
ammonium or sodium acetate, sodium succinate, sodium and/or potassium phosphate and
citrate-phosphate, where are employed as non-reducing sugars saccharose or trehalose;
as amino acids, glycine, histidine or leucine; as surfactants, polysorbate 20 or polysorbate
80 and as stabilizer polymer, polyethylene glycol or dextran or hydroxyethyl starch.
4. Freeze-dried stablilized pharmaceutical formulations as described in claim 3, where
is employed a buffer solution in concentration range between 10 and 20 mM; where the
saccharose or trehalose are employed in a concentration range between 5 and 100 mg/mL;
where the glycine, histidine or leucine are employed in a concentration range between
1 and 20 mg/mL; where the polysorbate is employed in a concentration range between
0.01 and 1 mg/mL and where the polyethylene glycol, dextran or hydroxyethyl starch
are employed in concentration range between 5 and 50 mg/mL.
5. Freeze-dried stabilized pharmaceutical formulation as describe in claim 2, composed
of IFN gamma in a concentration range between 5.6 x 108 IU and 1.4 x 108 IU and IFN alpha 2 in a concentration range between 6.8 x 108 IU y 1.7 x 108 IU, potassium di-hydrogen phosphate 0.0802 g, di-hydrated di-sodium hydrogen phosphate
0.249 g, saccharose 4 g, , glycine 0.8 g, Tween 20, 0.03 g, , polyethylene glycol
6000 1 g, and water for injection in an amount sufficient for 100 mL and for 0.5 mL,
1 mL, 5 mL y 10 mL, in the respective equivalents proportions.
6. Freeze-dried stabilized pharmaceutical formulation as describe in claim 2, composed
of IFN gamma in a concentration range between 2.0 x 108 IU and 0.5 x 108 IU and IFN alpha 2 in a concentration range between 12.0 x 108 IU and 3.0 x 108 IU, potassium di-hydrogen phosphate 0.0802 g, di-hydrated di-sodium hydrogen phosphate
0.249 g, saccharose 4 g, glycine 0.8 g, Tween 20 0.03 g, polyethylene glycol 6000
1 g, and water for injection in an amount sufficient for 100 mL and for 0.5 mL, 1
mL, 5 mL y 10 mL, in the respective equivalents proportions.
7. Freeze-dried stabilized pharmaceutical formulation as describe in claim 2, composed
of IFN gamma in a concentration range between 4.0 x 108 IU and 1.0 x 108 IU and IFN alpha 2 in a concentration range between 80 x 108 IU and 20 x 108 IU, potassium di-hydrogen phosphate 0.0802 g, di-hydrated di-sodium hydrogen phosphate
0.249 g, saccharose 4 g, glycine 0.8 g, Tween 20 0.03 g, polyethylene glycol 6000
1 g, and water for injection in an amount sufficient for 100 mL and for 0.5 mL, 1
mL, 5 mL y 10 mL, in the respective equivalents proportions.
8. Liquid stabilized pharmaceutical formulations as described in claim 1, that comprise
a buffer solution and at least a component selected from non-reducing sugar compounds,
amino acids, surfactants, stabilizer polymer, chelating/anti-oxidant compounds and
isotonizing agents, wherein the liquid formulation employs a water solvent that can
optionally contain preservants as a mixture of methyl-paraben or propyl-paraben.
9. Liquid stabilized pharmaceutical formulations as described in claim 8, where is employed
a buffer solution with the capacity to maintain the pH between 4.9 and 6.5, as ammonium
or sodium acetate, sodium succinate, sodium and/or potassium phosphate and citrate-phosphate,
and where is used as surfactant, polysorbate 20 or polysorbate 80; as antioxidant/chelating
EDTA or acetyl-cistein; as amino acids, histidine, L-arginine, L-alanine, glycine
or lysine; as stabilizer polymer, hydroxyethyl starch or dextran and as isotonizing
agent, sodium choride, potassium choride, propilenglycol, manitol, glycerol, saccharose
or trehalose.
10. Liquid stabilized pharmaceutical formulations as described in claim 9, where is employed
a buffer solution in a concentration range between 10 and 100 mM; where the polysorbate
is employed in a concentration range between 0.01 and 1 mg/mL; where the EDTA or acetyl-cysteine
are employed in a concentration range between 0.01 and 1 mg/mL; the histidine, L-arginine,
L-alanine, glycine, or lysine are employed in a concentration range between 1 and
20 mg/mL; hydroxyethyl starch and dextran are employed in a concentration range between
5 and 50 mg/mL and where the isotonizing agents are in sufficient amount to make the
solution isotonic.
11. Liquid stabilized pharmaceutical formulation as described in claim 9, composed of
IFN gamma in a concentration range between 5.6 x 108IU and 1.4 x 108 IU and IFN alpha 2 in a concentration range between 6.8 x 108 IU and 1.7 x 108 IU, sodium acetate 0.708 g, acetic acid 0.079 mL, Tween 20 0.01 g, manitol 5 g and
water for injection an amount sufficient for 100 mL and for 0.5 mL, 1 mL, 5 mL and
10 mL in a respective equivalent proportions.
12. Liquid stabilized pharmaceutical formulation as described in claim 9, composed of
IFN gamma in a concentration range between 2.0 x 108 IU and 0.5 x 108 IU and IFN alpha 2 in a concentration range between 12.0 x 108 IU and 3.0 x 108 IU, sodium acetate 0.708 g, acetic acid 0.079 mL, Tween 20 0.01 g, manitol 5 g and
water for injection an amount sufficient for 100 mL and for 0.5 mL, 1 mL, 5 mL and
10 mL in a respective equivalent proportions.
13. Liquid stabilized pharmaceutical formulation as described claim 9, composed of IFN
gamma in a concentration range between 4.0 x 108 IU and 1.0 x 108 IU and IFN alpha 2 in a concentration range between 80 x 108 IU and 20 x 108 IU sodium acetate 0.708 g, acetic acid 0.079 mL, Tween 20 0.01 g, manitol 5 g and
water for injection an amount sufficient for 100 mL and for 0.5 mL, 1 mL, 5 mL and
10 mL in a respective equivalent proportions.
14. Stabilized pharmaceutical formulations as described in any one of claims 1 to 13,
for the treatment of malignant or benign solid tumors, wherein the formulation is
employed independently or in a combination with chemotherapy, radiotherapy or the
combinations of both.
15. Stabilized pharmaceutical formulations as described in claim 14 for the treatment
of laryngeal carcinoma, laryngeal papilomatosis, acute myeloid leukemia, chronic myeloid
leukemia, lymphocytic acute leukemia, chronic lymphocytic leukemia, T cell leukemias,
B cell leukemias, central nervous system lymphoma, lipoma, epidermoid cyst, intradermal
cyst, liposarcoma, neurofibroma, sebaceous hyperplasia, cavernous hemangioma, hepatocellular
adenoma, focal nodular hyperplasia, astrocytoma, multiform glioblastoma, ependymoma,
ganglioneuroma, juvenile pilocityc astrocytoma, mixed gliomas, oligodendrogliomas,
optic nerve glioma, cordoma, craniopharyngioma, medulloblastoma; meningioma, pineal
tumor, pituitary adenoma, primitive neuroectodermal tumors, acoustic neuroma, vascular
tumors, meningeal carcinomatosis, neurofibromatosis, brain pseudotumors, tuberose
sclerosis, metastasic brain tumors, cherry angiomas, sebaceous gland hyperplasia,
basal cell carcinoma, squamous cell carcinoma, dermatofibroma, piogenic granuloma,
dermal nevus, seboreic keratosis, actinic keratosis.
16. Stabilized pharmaceutical formulations as described in any one of claims 1 to 13,
for the treatment of proliferative events as fibrosis, dysplasias, and hyperplasias.
17. Stabilized pharmaceutical formulations as described in any one of claims 1 to 13 for
intramuscular, intratumoral and perilesional application.
18. Topical stabilized pharmaceutical formulations as described in claim 1, comprising
recombinant IFN gamma in a concentration range between 0.32 x 106 IU and 0.08 x 106 IU and recombinant IFN alpha 2 in a concentration range between 2.0 x 106 IU and 0.5 x 106 IU per semisolid gram.
19. Stabilized pharmaceutical cream as described in claim 18, which comprise 2.2% IFN
gamma, 0.58% IFN alpha 2b, 4% cetyl alcohol, 10% petroleum jelly, 2% Tween 60, 0.2%
methylparaben and propylparaben and 81.2% distilled water csp.
20. Stabilized unguent as described in claim 18, which comprise 2.2% IFN gamma, 0.58%
IFN alpha 2, 60% white solid petrolatum, 10% heavy liquid petrolatum, 3% Span 20,
0.2% methylparaben and propylparaben and 24.02 % distilled water, csp.
21. Stabilized pharmaceutical formulation gel as described in claim 18, which comprises
2.2% IFN gamma, 0.58% IFN alpha 2, 0.5% Carbopol 940, 0.2% of methylparaben and propylparaben,
0.2% sodium hydroxide, 0.01 % calcium disodium ethylene diamine tetra-acetate, 2%
ethanol and 84.31 % distilled water, csp.
22. Topical stabilized pharmaceutical formulations as described in any one of claims 18
to 21, for the treatment of malignant or benign solid tumor of the skin or mucosa
employed independently or combined with chemotherapy, radiotherapy or combinations
of both.
23. Topical stabilized pharmaceutical formulation as described claim 22, for the treatment
of lipoma, epidermoid cyst, intradermal cyst, liposarcoma, neurofibroma, sebaceous
hyperplasia, hemangiomas, focal nodular hyperplasia, ependymoma, ganglioneuroma, juvenile
pilocityc astrocitoma, meningioma, pineal tumor, pituitary adenoma, vascular tumor,
meningeal carcinomatosis, neurofibromatosis, cherry angioma, sebaceous gland hyperplasia,
basal cell carcinoma, squamous cell carcinoma, dermatofibroma, piogenci granuloma,
dermal nevus, seborreic keratosis, actinic keratosis and condyloma.
24. A pharmaceutical kit comprising the pharmaceutical formulation as describe in any
one of claims 5 to 7 and 11 to 13, wherein said kit contains one or more vials of
recombinant IFN gamma, with the corresponding vials of recombinant IFN alpha 2 for
mixing the containment of IFNs vials in a way that the IFN gamma and IFN alpha concentrations
defined in the said claim is maintained and where additional the pharmaceutical kit
contain vials with water for injection, syringes and sufficient needles to carry out
the mixture of the IFNs and the application of the mix to patients.
REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
Patent documents cited in the description
- US5830455A [0002]
- US5028422A [0002]
- US5256410A [0002]
- US5024833A [0002]
- US5444064A [0002]
- US5814640A [0002]
- US5002764A [0002]
- EP0107498A [0006]
- US5190751A [0009]
- EP010749A [0009]
- US4499072A [0011]
- US5002879A [0011]
- US5503828A [0014]
- US4503035A [0014]
- US5762923A [0015]
- US4847079A [0015]
- US4675184A [0015]
- US5236707A [0015]
- US5431909A [0015]
- US4496537A [0016]
- US5935566A [0017]
- US0170207A [0018]
- WO8904177A [0019]
- US4895716A [0019]
- US5676942A [0020]
Non-patent literature cited in the description
- BORDEN E. et al.Progress in Hematology.19810000vol. XII, 299-339 [0002]
- DAHL H.Human interferon and cell growth inhibition. VII. Reversibility of interferon activitiesJ Interferon Animal, 1983, vol. 3, 327-332 [0002]
- WILLSON J.K.V.BITTNER G. et al.Antiproliferative activity of human interferons against ovarian cancer cells grown in human tumor stem cell assayJ Interferon Animal, 1984, vol. 4, 441-447 [0002]
- HU R.GAN Y. et al.Evidence for multiple binding sites for several components of human lymphoblastoid interferon-alphaJ Biol Chem, 1993, vol. 268, 12591-12595 [0002]
- QUESADA JR.TALPAZ M. et al.Clinical toxicity of interferons in cancer patients: to reviewJ Clin Oncol, 1986, vol. 4, 234-243 [0002]
- Clinical use of interferons. Solid tumors INTERFERONSTRANDER H.OBERG K.Principles and Medical Applications19920000533-561 [0003]
- LANE H. C.Interferon-alpha in patients with asymptomatic human immunodeficiency virus (HIV) infection. A randomized, placebo-controlled trialAnnals of internal Medicate, 1990, vol. 112, 805-811 [0004]
- TREDGET EEWANG R. et al.Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivoJ Interferon Cytokine Animal, 2000, vol. 20, 143-151 [0005]
- FUJII A.YUI-IN K. et al.Preliminary results of the alternating administration of natural interferon-alpha and recombinant interferon-gamma for metastasic renal cell carcinomaBJU Int., 1999, vol. 84, 399-404 [0007]
- HUBBELL H.R.CRAFT J.TO. et al.Synergistic antiproliferative effect of recombinant alpha-interferons with recombinant gamma-interferonJ Biol Response Mod, 1987, vol. 6, 141-153 [0007]
- MARTYRE M. C.BEAUPAIN R. et al.Potentiation of antiproliferative activity by mix of human recombinant IFN-alpha 2 and -gamma on growth of human cancer nodules maintained in continuous organotypic cultureEur J Cancer Clin Oncol, 1987, vol. 23, 917-920 [0007]
- HAND A.PELIN K. et al.Interferon-alpha and interferon-gamma combined with chemotherapy: in vitro sensitivity studies in non-small cell lung-cancer cell linesAnticancer Drugs, 1993, vol. 4, 365-368 [0007]
- MIZUKOSHI E.KANEKO S. et al.Up-regulation of type I interferon receptor by IFN-gammaJ Interferon Cytokine Animal, 1999, vol. 19, 1019-1023 [0008]
- OKUSE C.RINAUDO J. A. et al.Enhancement of antiviral activity against hepatitis C virus in vitro by interferon combination therapyAntiviral Animal, 2005, vol. 65, 23-34 [0008]
- SASAGAWA T.HLAING M. et al.Synergistic induction of apoptosis in murine hepatoma Hepa1-6 cells by IFN-GAMMA and TNF-alphaBiochem Biophys Common Animal, 2000, vol. 272, 674-680 [0008]
- CZARNIECKI C. W.FENNIE C. W. et al.Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferonsJ Virol., 1984, vol. 49, 490-496 [0009]
- KNUPFER M. M.KNUPFER H. et al.Interferon-gamma inhibits growth and migration of A172 human glioblastoma cellsAnticancer Animal, 2001, vol. 21, 3989-3994 [0011]
- MAHALEY M. S.BERTSCH L., JR. et al.Systemic gamma-interferon therapy for recurrent gliomasJ Neurosurg, 1988, vol. 69, 826-829 [0011]
- REDDY P. G. et al.Systemic gamma-interferon therapy for recurrent gliomasJ Natl Cancer Inst, 1991, vol. 83, 1307-1315 [0011]
- BUCKNER J. C.BURCH P. A. et al.Phase II trial of recombinant interferon-alpha-2a and eflornithine in patients with recurrent gliomaJ Neurooncol., 1998, vol. 36, 65-70 [0011]
- CHANG S. M.BARKER F. G. et al.High dose oral tamoxifen and subcutaneous interferon alpha-2a for recurrent gliomaJ Neurooncol, 1998, vol. 37, 169-176 [0011]
- DJORDJEVIC V.MILOVANOVIC J. et al.Radical surgery of the malignant laryngeal tumorsMinutes Chir Lugosl, 2004, vol. 51, 31-35 [0012]
- CHEN W.GUO X. et al.Long-term follow-up observation of clinical therapy for laryngeal carcinoma recurrence and cervical metastasisLin Chuang Er Bi Yan Hou Ke Za Zhi., 2004, vol. 18, 536-537 [0012]
- MANTZ C. A.VOKES E. E.Sequential induction chemotherapy and concomitant chemoradiotherapy in the management of locoregionally advanced laryngeal cancerAnn Oncol, 2001, vol. 12, 343-347 [0013]
- CLAYMAN G. L.YOUNG G. et al.Detection of regulatory factors of lymphokine-activated killer cell activity in head and neck cancer patients treated with interleukin-2 and interferon alphaAnn Otol Rhinol Laryngol, 1992, vol. 101, 909-915 [0013]
- PEREA S. et al.Minutes Cient Venez, 1993, vol. 44, 22-27 [0053]
- H. SANTANA.ESPINO Y. et al.A sandwich-type enzyme-linked immunosorbent assay for the analysis of recombinant human interferon α-2bBiotechnology Techniques, 1999, vol. 13, 341-346 [0059]
- BOUYÓN R.SANTANA H. et al.Development and validation of an enzyme-linked immunosorbent assay (ELISA) for recombinant human gamma interferonJournal of Immunoassay and Immunochemistry, 2003, vol. 24, 1-10 [0059]
- FERRERO J.OCHAGAVIA ME. et al.Titulación de la actividad antiviral del interferón utilizando el sistema de equipos SUMABiotecnología Aplicada, 1994, vol. 11, 34-42 [0059]
- FERRERO J.DUANY L et al.Nuevo programa de cálculo, cuantificación de la actividad antiviral de interferones mediante la inhibición del efecto citopatogénico utilizando el sistema de equipos SUMABiotecnología Aplicada, 1997, vol. 14, 267-269 [0059]